Our goal is to define the roles of Pten phosphorylation remodeling in hematopoietic stem cell (HSC) regulation. Pten, a tumor suppressor, has both lipid and protein phosphatase activities that inhibit PI3K/Akt and Fak-MAPK signaling respectively. It regulates many aspects of cell behavior, including proliferation, survival, adhesion and migration.1-4 The phosphatase activities of Pten are regulated by its c-terminal tail phosphorylation.5, 6 In addition, Pten many also have some phosphatase-independent functions. Many of Pten's biological functions are dependent upon protein-protein interactions which are mediated by its PDZ-motif.7 Knockout of Pten in mouse hematopoietic tissues results in abnormal activation of PI3K/Akt and Src signaling, which leads to uncontrolled HSC activation (G0 to G1 transition) and mobilization, followed by HSC decline. These mice develop myeloproliferative disorder (MPD) followed by acute myeloid/T lymphoid leukemia. Although Pten mutations are not commonly found in hematopoietic malignancies, including leukemia, p-Pten (the phosphorylated form of Pten) levels are increased in the abnormal blasts of most leukemic patients' bone marrow samples. Phosphorylation of Pten's c-terminal tail (ser380, thr382, and thr383) leads to a conformation change which may result in the blocking of its ability to bind to other partner proteins, the reduction of Pten phosphatase activity, and/or the alteration of the lifespan of the Pten protein. Our recent studies have suggested that the phosphorylation of Pten's c-terminal tail may not affect its lipid phosphatase activity but significantly compromises its protein phosphatase activity. The non-phosphorylated form of Pten (non-p-Pten) inhibits Src/Fak/p38 activity, thus repressing cell migration/invasiveness and inducing cell:cell contact inhibition of growth. p-Pten might have a dominant-negative function which induces cell-contact-related Src/Fak/p38 activation. We found that non-p-Pten is expressed in HSCs, while p- Pten levels are increased when HSCs enter the cell cycle; both of these events correspond to increased p- Src, p-Fak and p-p38 levels. Transduced over-expression of non-p-Pten preserves HSCs in a bone marrow niche-dependent manner, whereas transduced over-expression of p-Pten induces HSC/progenitors (HSC/Ps, from wild-type mice which have endogenous Pten expression) to differentiate to myeloid precursors. We propose that non-p-Pten maintains HSC quiescence and self-renewal ability through inducing cell:cell (HSCs and niche cells) contact-induced inhibition of growth by inhibiting Src/Fak/p38 signaling activities, whereas Pten's c-terminal phosphorylation alters its ability to bind to its partners and compromises its protein phosphatase activity. p-Pten promotes opposite functions to these through inducing cell:cell contact-related Src/Fak/p38 signaling. These studies will provide insights into how quiescent HSCs become activated and expand in number, and how we might be able to induce activated HSCs to revert back into quiescence in order to enhance their engraftment ability. This should greatly help our ability to expand HSCs in vitro and hence improve the outcome of clinical stem cell transplantation. It might be also help us to understand the nature of Pten c-terminal phosphorylation in leukemogenesis.